Proteins containing the Paired Box (Pax) DNA binding domain are key regulators of early developmental pattern formation in a wide variety of metazoan species. The role of these genes has chiefly been inferred from loss of function mutations, which have been shown to be the cause of a number of developmental anomalies in man, mouse, Drosophila and Caenorhabditis elegans. Despite the fundamental role that these genes appear to have in metazoan development, relatively little is yet known of the mechanisms by which their transcription is regulated. The work proposed here is directed at understanding the transcriptional regulation of Pax proteins and exploits two aspects of the sea urchin developmental model: (1) the ability to isolate immense quantities of embryonic nuclei for transcription factor isolation, and (2) the practicality of testing reporter gene constructs in large numbers of developing embryos. These tools will be used to: (1) map regulatory binding sites in a sea urchin Pax homolog. (2) determine the function of these sites in transcriptional control, and (3) identify and characterize upstream regulatory genes that control Pax expression during early development. The ultimate goal of the proposed research is to define not only the immediate cis-binding factors and mechanisms that allow for the controlled expression of Pax genes.but to characterize the upstream trans-cis regulatory networks that lead eventually to their controlled transcription. In view of the apparent extreme conservation of function that has been observed for a number of Pax genes in species ranging from Drosophila to humans. The information obtained from the proposed research is likely to have broad application towards understanding the control of Pax expression in vertebrates.